TUBB1 Monoclonal Antibody

What is TUBB1 and what is its biological significance?

TUBB1 (Tubulin Beta 1 Class VI) encodes for a specific member of the β-tubulin protein family. β-tubulins heterodimerize with α-tubulins to form the fundamental building blocks of microtubules, which are essential cytoskeletal structures in eukaryotic cells. While initially characterized as specifically expressed in platelets and megakaryocytes, recent research has demonstrated TUBB1 expression in thyroid tissue during development and in adulthood in both humans and mice . The protein plays crucial roles in:

• Platelet formation and function - affecting proplatelet formation and release

• Thyroid gland development - influencing thyroid migration during embryogenesis

• Thyroid hormone secretion - contributing to proper endocrine function

This expanded understanding of TUBB1 expression patterns has revealed unexpected roles in thyroid physiology beyond its known functions in platelet biology .

How is TUBB1 expression regulated during development and in different tissues?

TUBB1 exhibits a tissue-specific and developmentally regulated expression pattern. In human thyroid tissue, TUBB1 mRNA is expressed at gestational weeks 8, 10, and 12, and persists into adulthood . Similarly, in mouse thyroid tissue, Tubb1 is expressed at embryonic day 13.5 (E13.5) and shows stronger expression at E15.5, E17.5, and adulthood .

Cell sorting experiments in mouse thyroid tissue have demonstrated that Tubb1 is expressed in:

• EpCAM-positive epithelial cell populations containing thyrocytes (at E17.5 and in adults)

• CD41-positive megakaryocyte lineage cells (as expected based on previous knowledge)

Immunohistochemistry of human thyroid tissue has confirmed β1-tubulin protein expression in the cytoplasm of thyroglobulin-producing thyrocytes at gestational week 12 .

What are the recommended applications and dilutions for TUBB1 monoclonal antibody in different experimental techniques?

TUBB1 monoclonal antibody can be utilized across multiple experimental platforms with the following recommended dilutions:

The optimal working dilution should be determined empirically by each laboratory based on their specific experimental conditions. The antibody demonstrates reactivity with human, mouse, and rat TUBB1 proteins .

How should TUBB1 monoclonal antibody be stored and handled to maintain its effectiveness?

For optimal antibody performance and longevity, follow these storage and handling guidelines:

• Long-term storage: Store at -20°C for up to one year in the buffer provided (typically PBS pH 7.4 with 150mM NaCl, 0.02% sodium azide, and 50% glycerol) .

• Short-term/working storage: For frequent use over periods up to one month, store at 4°C to avoid repeated freeze-thaw cycles .

• After reconstitution: Store at 4°C for one month or aliquot and store frozen at -20°C for longer periods .

• Avoid repeated freeze-thaw cycles as these can degrade the antibody and reduce binding efficiency.

• Note that the product contains sodium azide, which is hazardous and should be handled by trained personnel with appropriate precautions .

For Western blot applications specifically, researchers should optimize protein loading, blocking conditions, and detection methods to achieve optimal signal-to-noise ratios.

How can TUBB1 monoclonal antibody be used to study thyroid development and pathology?

Recent discoveries linking TUBB1 mutations to thyroid dysgenesis and congenital hypothyroidism have opened new research avenues . Researchers can employ TUBB1 monoclonal antibody to:

• Characterize expression patterns during embryonic thyroid development using immunohistochemistry on tissue sections from different developmental stages.

• Analyze subcellular localization of TUBB1 in thyrocytes relative to other microtubule components using co-immunofluorescence.

• Examine expression changes in thyroid dysgenesis models, comparing wild-type versus mutant TUBB1 expression patterns.

• Investigate protein-protein interactions between TUBB1 and other tubulin subunits or microtubule-associated proteins in thyroid tissue using co-immunoprecipitation followed by Western blot analysis.

• Assess TUBB1 dynamics during thyroid cell migration by time-lapse imaging combined with immunofluorescence in appropriate cell culture models.

This approach has revealed that TUBB1 mutations can lead to non-functional α/β-tubulin dimers that cannot be incorporated into microtubules, disrupting microtubule integrity and impairing thyroid migration and thyroid hormone secretion .

What are the methodological considerations when studying the dual role of TUBB1 in platelet and thyroid biology?

When investigating TUBB1's dual role in megakaryocyte/platelet function and thyroid development, researchers should consider:

• Cell type-specific effects: Design experiments that can distinguish between cell-autonomous effects in thyrocytes versus indirect effects mediated through platelet function.

• Temporal dynamics: Employ developmental time-course analyses using the antibody to track expression changes at critical developmental windows.

• Mutation-specific effects: Three distinct TUBB1 mutations (c.35delG, c.318C>G, and c.479C>T) have been identified in patients with thyroid dysgenesis . Each mutation should be assessed separately as they may affect protein function differently despite all preventing incorporation into microtubules.

• Compensatory mechanisms: In Tubb1 knockout mice, significant increases in expression levels of other β-tubulin isoforms (Tubb2a, Tubb5, Tubb2b, and Tubb3) occur in the thyroid, potentially compensating for Tubb1 loss . Western blot analysis with antibodies against multiple tubulin isoforms can help characterize these compensatory changes.

• Functional readouts: Combine morphological analyses with functional assays measuring thyroid hormone production and platelet aggregation to comprehensively assess the impact of TUBB1 alterations.

How can researchers address specificity concerns when working with tubulin antibodies?

The tubulin protein family contains multiple highly conserved isoforms that can create specificity challenges. To ensure TUBB1-specific detection:

• Validate antibody specificity using positive controls (platelets or megakaryocytes) and negative controls (tissues or cells with confirmed absence of TUBB1 expression).

• Perform knockout validation using Tubb1-knockout mouse tissues as negative controls if available.

• Consider cross-reactivity with other β-tubulin isoforms, especially since the three TUBB1 mutations identified in patients with thyroid dysgenesis affect amino acids that are strictly conserved across species and across all β-tubulins .

• Use multiple antibody clones targeting different TUBB1 epitopes to confirm results.

• Employ complementary techniques such as RNA in situ hybridization or RT-PCR to correlate protein and mRNA expression patterns.

• Design appropriate blocking experiments to confirm signal specificity, particularly when working with tissues known to express multiple tubulin isoforms.

What are common pitfalls in interpreting TUBB1 expression data in developmental studies?

When studying TUBB1 expression during development, researchers should be aware of:

• Developmental timing: Expression levels change significantly throughout development. For example, in mouse thyroid tissue, Tubb1 shows stronger expression at E15.5, E17.5, and adulthood compared to E13.5 . Ensure adequate temporal sampling.

• Cell heterogeneity: Thyroid tissue contains multiple cell types. Use cell sorting based on markers such as EpCAM (epithelial cells), Pecam (endothelial cells), Pdgfra (fibroblasts), and CD45 (leukocytes) to accurately determine cell type-specific expression .

• Signal interpretation: Distinguish between specific TUBB1 signals and background staining in immunohistochemistry by including appropriate controls and optimizing antibody concentrations.

• Compensatory mechanisms: Knockout models may trigger upregulation of other tubulin isoforms that can mask phenotypes. Western blot analysis in Tubb1-knockout mice revealed increased expression of Tubb2a, Tubb5, Tubb2b, and Tubb3, as well as decreased expression of Tuba3 and Tuba4 .

• Species differences: While TUBB1 functions are largely conserved across species, some species-specific differences in expression patterns or functional requirements may exist.

How have recent discoveries about TUBB1 mutations expanded our understanding of rare pediatric diseases?

The identification of TUBB1 mutations in patients with thyroid dysgenesis has significantly expanded the spectrum of rare pediatric diseases associated with tubulin-coding gene mutations . Research findings include:

• In a cohort study of 270 patients with congenital hypothyroidism and thyroid dysgenesis, 1.1% had TUBB1 mutations .

• Three distinct TUBB1 mutations were identified in three independent families:

  • c.35delG mutation (family F3)

  • c.318C>G mutation (family F2)

  • c.479C>T mutation (p.P160L, rs759117911) (family F1)

• Statistical analysis showed significant enrichment of rare TUBB1 variants in patients with thyroid dysgenesis compared to controls (5.2% vs. 2%, p=0.0227) .

• All three mutations affect highly conserved amino acids and are located in the N-terminal domain needed for GTP activity. Two mutations (c.318C>G and c.35delG) create premature stop codons that remove domains required for microtubule-associated protein binding .

• These findings connect thyroid development disorders with platelet abnormalities, suggesting potential screening opportunities for patients with one phenotype to identify the other.

What methodological approaches can researchers use to investigate the molecular mechanisms by which TUBB1 mutations affect microtubule dynamics?

To elucidate how TUBB1 mutations impact microtubule assembly and function, researchers can employ:

• In vitro tubulin polymerization assays: Compare microtubule assembly kinetics using purified wild-type versus mutant TUBB1 proteins.

• Live-cell imaging: Visualize microtubule dynamics in cells expressing wild-type or mutant TUBB1 tagged with fluorescent proteins.

• Structural biology approaches: Use X-ray crystallography or cryo-electron microscopy to determine how mutations affect the three-dimensional structure of TUBB1 and its interaction with α-tubulin.

• Immunoprecipitation studies: Identify altered protein-protein interactions between mutant TUBB1 and microtubule-associated proteins or other tubulin subunits.

• Functional rescue experiments: Test whether wild-type TUBB1 can rescue phenotypes in cells or organisms expressing mutant TUBB1.

• Computational modeling: Simulate the effects of specific mutations on tubulin dimer formation and microtubule polymerization.

These approaches have revealed that TUBB1 mutations can lead to non-functional α/β-tubulin dimers that fail to incorporate into microtubules, disrupting cytoskeletal integrity in both thyroid cells and platelets .